Treatment of protein and peptide materials to form amide linkages



, ture.

United States Patent .0

3,098,693 TREATMENT OF PROTEIN AND PEPTIDE MATE- RIALS TO FORM AMIDELINKAGES John C. Sheehan, Cambridge, Mass, assignor to Arthur D. Little,Inc., Cambridge, Mass., a corporation of Massachusetts No Drawing.Continuation of application Ser. No. 766,406, Oct. 10, 1956, now PatentNo. 2,938,892, dated May 31, 1960. This application May 27, 1960, 1 Ser.No. 32,112 15 Claims. (Cl. 8127.6)

The present invention relates to the formation of peptide and otheramide linkages, to the chemical agents which facilitate the formation ofsuch linkages and to the treatment of protein and peptide materials withsuch agents.

This application is a continuation of United States application SerialNo. 76 6, 406, filed October 10, 1958, now U.S. Patent 2,938,892 whichis a continuation-in-part of United States application Serial No.641,853, file-d February 2 5, 1957, which, in turn, is acontinuation-impart of Serial No. 551,531, filed December 7, 1955, forProdnot and Process, the latter two now abandoned.

It has now been found that peptides and other amide linkages may beformed readily in the presence of an aliphatic carbodiimide. Thealiphatic carbodiimides of the present invention have the generalstructure in which at least one aliphatic carbon atom is attached to oneof the nitrogen atoms of the N=O=N struc- Such aliphatic radicals may becyclohexyl, ethyl, n-propyl, isopropyl, n butyl, sec-butyl, iso-butyl,tertbutyl, amyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,benzyl, alphaand beta-phenylethyl, ethyl morpholinyl, diethylaminoethyl,ethylpiperidyl, alpha-, betaand gamma-methylor ethylpyridyl.

The carbodiimides of the present invention may be prepared in accordancewith the general method of E. Schmidt, F. Hitzler and E. Lahde, Ben, 71,1933 (1938), from the corresponding thioureas by oxidation with mercuricoxide in acetone. The thioureas may be prepared from the correspondingamine by reaction with carbon disulfide in the case of symmetricalthioureas. The unsymmetrical thioureas may be prepared by interaction ofan amine with an isothiocyanate. The carbodiimides are also preparablefrom the corresponding ureas, and are regeneratable after use, thuspermitting recycling of the condensation agent, following the procedureof Amiard, G. and Heymes, R., Bull. Soc. Chim. France, 1360 (1956).

Of the carbodiimides of the present invention those most preferred arethe water-soluble carbodiimides since the most useful applications ofthe process involve the use of aqueous media either during amideformation, or in the purification of the amide or in the separation ofthe resulting urea which may, if desired, be regenerated to carbodiimideand reused. Carbodiimides bearing tertiary amino groups may be renderedsoluble in water by neutralizing said amino group with an acid capableof forming a substantially neutral salt with the tertiary amine. Suchacids include the hydrohalic group, hydrogen chloride, hydrogen bromideand hydrogen iodide, sulfuric acid, sulfonic acids, nitric acid,phosphoric acid, phosphonic acids but 'ordinarly do not includecarboxylic acids since the latter place in the carbodiimide a carboxylgroup which provides a carboxyl group as the reactant in combining withthe amino group of the other reactant to form an amide linkage. Also,carbodiimides bearing tertiary amino groups may be rendered watersoluble by quaternization with methyl p-toluenesulfonate (methyl Ice.

tosyla-te), methyl bromide, methyl iodide, benzyl bromide, ethyl iodide,ethyl bromide, benzyl iodide, ethyl p-toluenesulfonate (ethyl tosylate),methyl sulfate, ethyl sulfate and the like. Other groups which renderthe carbodiimides water soluble include sulfonates, sulfates,phosphates, phosphonates, guanidinium, polyhydroxy, and polyethergroups. Where the aliphatic group attached to one of the nitrogen atomsof the N=C=N-- is of small molecular size or, in addition, contains apolar group, water solubility is imparted. Such groups are ethyl,dimethyl-aminoethyl, hydroxyethyl and the like. In rendering thecarbodiimide water soluble essentially neutral groups should beemployed. Sulfonic acid, carboxylic acid and quaternary ammonium basegroups are examples of groups that are often too acidic or basic to beemployed directly in the carbodiimides of the present invention.

As is evident to those skilled in the art the reaction shown in theaccompanying detailed examples involves the condensation of a primary orsecondary amino group and a carboxyl group with the elimination of amolecule of water and formation of an amide bond. This water is pickedup by the carbodiimide, preferably monocarbodiimide, resulting inconversion of the carbodiimide to the corresponding urea. This reactionis illustrated by the following general equation in which only thefunctional groups involved are shown: -COOH,-+HN+N=C=N- This reactionoccurs readliy under mild conditions of temperature with a high degreeof selectivity providing high yields and a minimum of side reactions andresultant by-products which would require extensive purification. Thereaction can be carried out in a wide variety of solvents ranging fromcompletely organic media such as dioxane, tetrahydrofuran, methylenechloride, acetonitrile, benzene, ethyl acetate, hexane, acetone,formamide, dimethyl formamide with and without lithium chloride toaqueous systems including water alone or with acetone, dioxane,tetrahydrofuran, acetonitrile and the like. With the instantwater-soluble carbodiimide condensing agent, it is also practical to useorganic solvent during the amide formation and aqueous systems forsubsequent purification and removal of the urea and unchangedcarbodiimide. The term water soluble as used in the specification andclaims with reference to carbodiimide refers to carbodiimides having asolubility in Water to an extent of at least 0.01% by weight andpreferably at least 0.1% by weight. Pressure has not been shown to be afactor. No catalysts have been found to offer any advantage, nor havethe concentration of reactants and carbodiimide been shown to becritical. Of course, it is best to employ equimolecular proportions ofreactants in the usual case although when carrying out the reaction inaqueous, alcoholic or other possibly reactive media it is advantageousto employ an excess of the carbodiimide reactant.

While the bonds formed in the examples are generally described aspeptide linkages it will be recognized that the carbodiimides of thepresent invention are equally applicable to the formation of amide bondsgenerally. The functional groups involved in the reaction are the samewhether protein, amino acids, peptides or non-peptide reactants areemployed and the linkage or grouping produced, CO-N, is the same. Thisis shown, for example, in the polymerization of adipieacid andhexamethylenediamine which proceeds through the formation of amide bondsusing the carbodiimides of the present invention under mild conditionsof temperature.

- In this invention, the utilization of a carbodiimide as condensingagent effects the formation of an amide by condensing an aminecontaining an amino group, capable of being acylated and having at leastone hydrogen attached to the amino nitrogen, with an organic carboxylicacid capable of forming amide bonds.

In accordance with the accompanying detailed examples, the reaction iseffected between carboxylic acids and ammonia, primary and secondaryamines; illustratively, primary aliphatic amines: such as, for example,methylamine, ethylamine, propylamine, butylamine, hexylamine,allylamine; secondary aliphatic amines, such as, dimethyl amine, diethylamine, dipropylamine, dibutylamine; substituted aliphatic amines, andaralkylamines; such as chloroethylamine, phenethylamine, benzylamine;aryl amines, aniline, alphanaphthylamine, betanaphthylamine; substitutedarylarnines, m-toluidine, p benzylaniline; secondary mixed aliphaticaromatic amines, such as N-allylaniline, benzylaniline; cyclic amines,piperidine, morpholine; heteracyclic amines, aminopyrimidine; diamines,butylenediamine, and ethylenediamine. The acids suitable for reaction inthe instant amide formation are organic carboxylic acids, capable offorming an amide with an amine, illustratively, aliphatic acids, acetic,propionic, butyric, lauric, oleic, glycollic, citric, lactic, glutaric,maleic, succinic, dimethylacetic, heptanoic, betacyclopentylpropionic,cholic, monochloracetic, dichloracetic, trichloracetic, acetoacetic,beta-acetylpropionic; aryl acids, benzoic, 2-meth ylbenzoic,Z-methoxybenzoic, salicyclic; parahydroxybenzoic, para-aminobenzoic,aralkylacid, betanaphthoic, phthalic; phenylacetic, cinnamic,betaphenylpropionic; heterocylic acids, furoic andbetapyridinecarboxylic.

case of using chrome alum, tannin, formaldehyde and like agents employedtoday. Such treatment with a carbodinnide is usually desirably precededby a treatment designed to make available an increased number ofcarboxyl groups. In most cases there are an adequate number of aminogroups available for the reaction so that no pretreatment for thispurpose is required. These are generally regarded as being providedprincipally by the terminal amino group of lysine. Such side aminegroups are supplied by glutamic and aspartic acid units in the proteinstructure. Swelling, softening, penetrating or surface active agents canbe employed to increase depth of effect.

Protein fibers such as hair, furs, silk, wools and the like may betreated with the carbodiimides of the present invention to impartdimensional stability, including resistance to shrinking, hardness,increased tensile strength and the like. This treatment of fibers canalso be preceded advantageously by such pretreatments as those describedabove in connection with hides.

Both hides and animal fibers can be treated in accordance with theinvention to increase Water repellency or resistance and wet strength.In such a case softening or swelling pretreatment offers littleadvantage but In the event that cysteine is selected for condensationwith amine by the instant process, it is desirable that the sulfhydrylgroup be first blocked by benzylation. As recognized in the art, wherereactive side chain substituents occur, and if desired, are to beprotected, they may be blocked, in the case of amino bycarbobenzoxylation, carboxyl by esterification to methyl or benzylesters, and hydroxyl by acetylation or carbobenzoxylation.

Reaction of amines and acids to form amides are of course most readilyeifected in the solution state, however either the amine or the acid maybe undissolved or in a different phase and reactions are effectedbetween undissolved solids functioning as acids and amines asillustrated by the treatment of wool, felt and leather with carbodiimidein accordance with this invention.

The carbodiimides of the present invention may be employed in thetreatment of protein materials such as animal hides, animal fibers,polypeptide fibers such as silk, wools, furs and hair, feathers, casein,zein, soy protein, gliadin, egg albumen, lactalbumen, gelatin and thelike. The use of the carbodiimides to form amide bonds has the generaleffect of increasing the molecular size of the protein treated Whetherit be that contained in hides, furs, feathers or the extracted proteinsuch as casein, albumens, zein, gelatin and the like. This increase inmolecular weight usually involves the formation of cross linkages,especially in the case of the fibrous proteins although to a lesserextent in the case of the globular proteins. In the treatment ofproteins with carbodiimide in accordance with this invention, it is nowpractical to avoid, if desired, the introduction of new reactants intothe protein and at the same time the proteins are modified but retaintheir characteristic antigenicity. Thus virus cultures and bacteria arecompletely inactivated by carbodiimide but show the characteristicantigenicity of their native protein upon injection into an animal.

Advantage can be taken of these general effects in the treatment ofhides to cause hardening or tanning.

In such a case no fragment is left in the hide. as in the treatment toprovide an increased number of free carboxyl groups at the surface ofthe hide or fiber is ad vantageous.

It is also possible as indicated above totreat extracted proteins suchas casein, zein, gliadin, soy protein, soy albumen, egg albumen,lactalbumen, gelatin and the like. In the case of casein the result isthe formation of high polymers useful in the production of molded orextruded plastic articles or in paints where enamel or hard surfaces aredesired. In the case of gelatin the carbodiimides of the presentinvention may be used to increase molecular weight or jellying powerusually expressed in terms of Bloom and they may also be used to hardenor otherwise modify the gelatin for photographic purposes, e.g., a 5%solution of gelatin in Water to which was added an amount of1-cyclohexyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemetho-p-toluene-sulfonate equal in weight to the gelatin set to a gel in60 seconds and after 13 minutes had hardened to the point where it couldnot be melted at steam-bath temperatures.

The gelatin of this invention is readily distinguishable from collagenor gelatin as heretofore known. Collagen is not water-soluble ascompared with gelatin. Both collagen and prior art gelatin containlysine in combined form as part of the protein, but with freeamino-nitrogen,

principally e-amino, analysis of about 0.64 percent. De aminated gelatinin which the amino groups have been removed and the amino acids thusaltered are known. The instant fully modified gelatin contains all ofits original amino acid content but with no added residues derived fromconventional hardening agents; such as, for example, aldehydes(formaldehyde or glyoxal) and metallic salts (chrome alum), and includea lysine content of about 4 percent, but with a Van Slyke freeamino-nitrogen analysis of less than about percent of the Van Slyke freeamino-nitrogen analysis of the corresponding untreated gelatin. Thismodified gelatin which has a Van Slyke free amino-nitrogen analysis ofabout 95 percent less than the Van Slyke free amino-nitrogen analysis ofthe corresponding untreated gelatin possesses increased viscosity inaqueous dispersion and is productive of higher melting point gels at aparticular concentration than is effected with ordinary gelatin. A lowerviscosity modified gelatin, without having a reduction in molecularweight or degradation, is effected by the modified gelatins having a VanSlyke free amino-nitrogen analysis of less than about 25 percent of theVan Slyke free aminonrtrogen analysis of the corresponding untreatedgelatin. Thus, substantially all of the free amino group content andcarboxyl content of a gelatin may be condensed by this invention or onlya relatively small portion of the bodiimides.

free amino group content and carboxyl content of a gelatin may becondensed. The proportion of the free amino group content and carboxylgroup content of a gelatin condensed may be controlled to obtain amodified gelatin having a particular melting point or other desiredproperty. The degree of modification of the gelatins modified by thisinvention may be determined, in addition to a Van Slyke freeamino-nitrogen content before and after modification, by reaction with2,4-dinitro-fluorobenzene (Sangers reagent) followed by total hydrolysisand measurement of free lysine. Thus, the modified gelatin having a VanSlyke free amino-nitrogen analysis of less than about 95 percent of theVan Slyke free amino-nitrogen analysis of the corresponding untreatedgelatin, when so tested, has an acylated lysine e-amino content of aboutpercent.

The carbodiimides of the present invention not only provide increasedmolecular weight by linear combination or end to end linkages toincrease the length of the protein molecule, but the carbodiimides alsoserve to cause cross linking between adjacent protein chains. It isparticularly advantageous that the carbodiimides provide both types oflinking since this, in turn, provides maximum effect with respect totanning hides, fixing fibers, hardening proteins and the like.

The carbodiimides of the present invention may be employed in thepreparation of pharmaceutical or biologically active compounds ormaterials. For example, gramicidin-S, which is a macrocyclic peptideantibiotic containlng ten amino acid units in a thirty-membered ring,could be prepared using the carbodiimides both in building up the tenamino acid unit chain and in the final cyclization. Similarly, peptidehormones such as those of the pituitary gland termed vasopressin andoxytocin may also be prepared using the carbodiimides of the presentinvention.

In accordance with this invention directed to the formation of amidelinkages as described in detail, those having particular utility are thewater-soluble aliphatic car- These and their preparation have beenexemplified. They are novel and possessed of especial ad- 3 vantage inthat the amide linkages may be formed in aqueous media and most aminoacids, peptides and pro teins are most soluble or compatible in suchmedia. Also, such carbodiimides result in water-soluble ureas which aremost easily removed from the reaction mixture or otherwise separatedfrom the product containing the newly formed amide linkage.Surprisingly, such carbodiimides dehydrate the carboxyl and amino groupsinvolved in the reaction and do so in aqueous media.

Detailed procedures for preparing carbodiimides typical of those of thepresent invention as set forth below:

EXAMPLE 1 1-Cycl0hexyl-3- [Z-Morpholinyl- (4) -Ethyl] Thz'ourea Asolution of 19.4 g. (0.138 mole) of cyclohexylisothiocyanate A. Skitaand H. Rolfes, Ben, 53B, 1242 (1920) and 18.0 g. (0.138 mole) ofN-(2-aminoethyl)- morpholine in 500 ml. of ether was heated under refluxfor minutes. On cooling, the crystalline thiourea which separatedamounted to 36.0 g. (96%), M.P. 128129 C.

A portion was rescrystallized from acetone-ligroin, M.P. 128-129 C.

EXAMPLE 2 1-Cyclohexyl-3- [2-M0rp'h0linyl-(4 -Ethyl] Carbodiimide Amixture of 40 g. (0.0147 mole) of 1-cyclohexyl-3-[2-morpholinyl-(4)-ethyl] thiourea and 6.0 g. of mercuric oxide (Merckyellow) in 50 ml. of acetone was heated (magnetic stirrer) under refluxfor 6 hours. The mercuric sulfide formed, was removed by filtration, asecond 6 g. portion of mercuric oxide was added, and the suspension washeated at reflux for another 6 hours. The reaction mixture was againfiltered and the filtrate concentrated 6 under reduced pressure. Theoily residue was evaporatively distilled at 140 C./0.2 mm; yield, 2.4 g.(70%). Arralysis.Calod. tor C H N O: C, 65.78; H, 9.77; N, 17.71. Found:C, 65.60; H, 9.59; N, 17.71.

EXAMPLE 3 1-Cycl0hexyl-3-[2-Morph0linyl-(4)-Ethyl]-Carbodiimide M etho-pToluenesulfonate EXAMPLE 4 1-Cycl0hexyl-3-(4-Diethylam inocyclohexyl)Thiourea After heating under reflux for 15 minutes a solution of 5.0 g.(0.029 mole) of N,N-diethyl-1,4-cyclohexyldiamine and 4.2 g. (0.03 mole)of cyclohexylisothiocyanate in 10 ml. of ether was evaporated underreduced pressure. Crystallization of the residue from warm 'ligroin andrecrystallization from an acetone-ligroin mixture aflorded 7.2 g. M.P.139-141 C., of the pure thiourea.

Analysis.Calcd. for C H N S: C, 65.59; H, 10.61; N, 13.50; S, 10.28.Found: C, 65.65; H, 10.66; N, 13.60; S, 10.36.

EXAMPLE 5 1-Cyclohexyl-3-(4-Diethylaminocyclohexyl) Carbodiimide Asuspension of 2.0 g. of mercuric oxide in 20 ml. of acetone containing0.8 g. (0.003 mole) of 1-cyclohexyl-3- (4-diethylaminocyclohexyl)thiourea was refluxed for 20 hours. The mercuric sulfide formed wasremoved by filtration, the filtrate was concentrated under reducedpressure, and the residue was evaporatively distilled at 140 C./0.03mm., 0.6 g. (84%).

Analysis.Calcd. for C H N C, 75.59; H, 11.26; N, 15.15. Found: C, 73.19;H, 11.30; N, 15.29.

EXAMPLE 6 1 -Cyclohexyl-3- (4-D iethylwmi nocyclohexyl Carbodiimid e Metho-p Tol uenesul fanate A solution of 0.3 g. (1.1 mmole) of1-cyclohexyl-3-(4- diethylaminocyclohexyl) carbodiimide and 0.21 g. (1.1mmole) of methyl p-toluenesulfonate in 6 ml. of benzene (sodium dried)was heated under reflux on a steam bath for 20 minutes. After storageovernight at room temperature, the solvent was evaporated to drynessunder reduced pressure. The hygroscopic residue, amounting to 0.51 g.(100% was analyzed without further purification.

Armlysis.-Calcd. for C H N O S: C, 64.78; H, 8.85; N, 9.07. Found: C,64.58; H, 8.71; N, 9.14.

EXAMPLE 7 1,3-Di-(4-Diethylamino cyclohexyl) T hiourea A solution of 2.3ml. (0.03 mole) of carbon disulfide in 4 cc. of methanol was addedslowly to a cold solution of 2.0 :g. (0.012 mole) ofN,N-diethyl-1,4-cyolohexyldiamine in 20 mi. of methanol. As the additionprogressed a solid separated. This suspension was heated under refluxfor 2 days during which time solution was effected. The reaction mixturewas evaporated to dryness under reduced pressure, and the residue wasrecrystallized twice from ligroin (B.P. -100 C.), yielding 0.8 g. (35%),M.P. 164165 C. V

' An.wlysis.Calcd. for C H N S: C, 65.97; H, 11.00; N, 14.55; S, 8.36.Found: C, 65.80; H, 11.22; N, 14.74; S, 8.33.

7 EXAMPLE 8 1,3-Di-(4-Diethylam inocyelohexyl) Carbodiimz'de A mixtureof 2.4 g. of mercuric oxide and 0.95 g. (2.5 mrnoles) of1,3-di-(4-diethylaminocyclohexyl) thiourea in 30 cc. of acetone washeated with stirring under reflux for 1 hour. The filtered reactionmixture was concentrated under reduced pressure and the residue wasevaporatively distilled at 180 C./0.03 mm., 0.575 g. (66%Analysis.-Calcd. for C H N C, 72.36; H, 11.57; N, 16.08. Found: C,72.25; H, 11.50; N, 16.34.

EXAMPLE 9 1 -Cyclohexyl-3- fi-Diethylaminoethyl) Carbodiimide A solutionof 7.0 g. (0.05 mole) of cyclohexyl-isothiocyanate A. Skita and H.Rolfes, Ber., 53B, 1242 (1920), and 5.8 g. (0.05 mole) ofN,N-diethylethylene diamine in ether was heated under reflux for 10minutes. The ether was evaporated to dryness under reduced pressure,leaving a yellow oil, weight 12.8 g.

A portion of the thiourea g.) was dissolved in 75 cc. of acetonecontaining 7.2 g. of mercuric oxide (Merck yellow). The reactionsolution was stirred and heated under reflux for 5 hours. The mercuricsulfide was removed by filtration and an additional 7.0 g. of mercuricoxide was added followed by an overnight reflux period. Afterevaporation of the solvent, the residual oil was purified by evaporationdistillation at 100 C./ 0.05 mm.; yield, 12 g.

Analysis.-Caled. for C H N C, 69.90; H, 11.28; N, 18.81. Found: C,69.91; H, 11.24; N, 18.75.

EXAMPLE 1-Ethyl-3- [2-M0rphl0linyl-(4) -Ethyl] T hioureaEthylisothiocyanate (25.0 g.; 0.287 mole) Was added slowly with stirringto a solution of 37.4 g. (0.287 mole) of aminoethylmorphol-ine (carbideand carbon) in 400 ml. of dry ether. The product crystallized from thesolution during the addition. After an additional 2 hours of stirring atroom temperature the product was collected. The yield was 56.0 g. (90%M.P. 94.5-96.0 C.

EXAMPLE 11 1-Ethyl-3- [Z-Morpholinyl-(4) -Ethyl] Carbodiimide A mixtureof 54.2 g. (0.25 mole) of 1-ethyl-3-[2-morpholinyl-(4)-ethyl] thioureaand 54.0 g. (0.25 mole) of freshly-dried mercuric oxide (Merck yellow)in 500 ml. of dry acetone was stirred at room temperature for 12 hours.The mercuric sulfide which formed was removed by filtration. Thisprocess was repeated with two 27.0-g. portions of mercuric oxide, thefinal mixture being treated with activated carbon before filtration. Thecolorless filtrate was concentrated under reduced pressure to a paleorange oil, 42.1 g. (92%).

Flash distillation through a short-path still (oil bath 135 C.; 0.2 mm.)gave 22.625 g. (50%) of colorless carbodiimide; 11 1.4944; d 1.0235.

EXAMPLE 12 1-Ethyl-3-[Z-Morpholinyl-( l)-Ethyl] Carbodiimia'e Metho-p-Tol uenesulfonate A mixture of the basic carbodiimide (3.27 g.;0.018 mole) and methyl p-toluenesulfonate (3.32 g.; 0.018 mole) washeated with constant swirling on a steam bath for 5 minutes. Theresulting viscous, orange oil was crystalvlized from acetone-benzene to4.2 g. (64%) of colorless quaternary carbodiimide, M.P. 9 1.5-92.5 C.

EXAMPLE 13 1-Ethyl-3-[2-M0rph0linyl-(4)-Ethyl] Carbodiimide Two grams(0.0098 mole) of 1-ethyl-3-[2-morpholinyl- (4)-ethyl] urea (from ethylisocyanate and the amine) and 3.82 g. (0.02 mole) of p-toluenesulfony-lchloride were mixed slowly with 5.5 ml. pyridine (reagent) in 20 ml. drymethylene chloride with stirring at 0 C. The resulting yellow solutionwas refluxed gently cfor a period of 4 hours with stirring.

The solution was then added slowly to 20 g. of ice and 2 g. of sodiumcarbonate. Additional sodium carbonate was added as the mixture wasadded to the ice, so that at all times the pH of the solution wassomewhat above 7. The final pH of the mixture was 8. The prodnot wasimmediately extracted with three 30-ml. portions of methylene chloride.This extract was filtered through sodium sulfate and dried over sodiumsulfate.

The solvent was removed by warming at 2 mm. Hg. The residual orange oilwas distilled with N ebullition at 1.5 mm, B.P. 10010*5 C., yield 0.37g. 20% o tthe colorless product, 1 ethyl 3 [2-morpholinyl-(4)-ethyl]carbodiimide.

EXAMPLE 14 1 -Ethyl-3- [2-M0rphl0linyl-(4) -Ethyl] CarbodiimideHydrochloride To -a cooled (0-5" C.) solution of the basic carbodiimide,l-ethyl-Zl-[2-morpholinyl-(4)-ethyl] carbodiimide (3.27 g.; 0.018 mole),in 20 ml. of anhydrous ether was added slowly 0.017 mole of hydrogenchloride in 10 ml. of ether. After removal of solvent the water-solublecarbodiimide salt, 1 ethyl 3 [2-morpholinyl-(4)-ethyl] carbodiimidehydrochloride, is used without further purification.

EXAMPLE 15 1-Ethyl-3-[2-M0rph0linyl-(4) -Ethyl] Carb odiimide Sulfate Toa cooled (0-5 C.) mixture of the basic carbodiimide,1-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimide (3.27 g.; 0.018 mole),and 20 ml. of water was added 0.017 mole of N-sulfuric acid. Thesolution was immediately lyophilized. The resulting semi-solid,1-ethyl-3- [2-morpholinyl-(4)-ethyl] carbodiimide sulfate, is used foramide condensations Without 'further purification. Alternatively, theaqueous solution prepared is used to effect the cross-linking ofgelatin.

EXAMPLE 16 1,3-Di-('y-Diethylaminopropyl) Carbodiimide Five grams ofl,3-di-('y-diethylaminopropyl) urea (from the corresponding amine andurea) and 9 g. of ptoluenesulfonyl chloride were mixed slowly with 12ml. of pyridine and 60 ml. of dry methylene chloride with stirring at 0C. After a reflux period of -6 hours the cooled solution was poured onto60 g. of ice and 7 g. of potassium carbonate. Addition-a1 potassiumcarbonate was added to maintain the p-H at 8. The product was extractedwith tour portions of methylene chloride and the dried (sodium sulfate)extract was concentrated under reduced pressure. The 1,3-di-('-diethylaminopropyl) carbodiimide can be used without furtherpurification. The yield is 25%.

EXAMPLE 17 1-Ethyl-3-('y-Dimethylaminopropyl) Carbodiimide Two grams ofl-ethyl-3-('y-dimethylaminopropyl) urea (from ethyl isocyanate andN,N-dirnethyl-1,3-propanediamine) and 3.5 g. of p-toluenesulfonylchloride were added slowly to 5 ml. of pyridine in 30 ml. of drymethylene chloride with stirring at 0 C. After a 4-hour reflux periodthe cooled solution was added slowly to a mixture of 20 g. of ice and 2g. of sodium carbonate. Additional sodium carbonate was added tomaintain the pH of the solution at 8. The layers were separated and theorganic extract was dried over sodium sulfate and the solvent underreduced pressure. The 1-ethy1-3-(y-dimethylarninopropyl) carbodiimidemay be used directly or may be purified by distillation at 1 mm. Theyield is 30%.

EXAMPLE 18 Phthaloylglycylglycine Ethyl Ester To a solution of 0.18 g.(0.865 mmole) of phthaloylglycine and 0.088 g. (0.865 m-inole) ofglycine ethyl ester in 6 ml. of water was added 0.4 g. (0.86 mmole) of 1cyclohexyl-B-(4-diethylaminocyclohexyl) carbodiimide methop-toluenesulf'onate. A solid began to separate after 10 minutes ofstirring and after 2 hours the solid, phthaloylglycylglycine ethylester, was collected, weight 0.18 g. (75%); M.P. 194195 C.

The aqueous phase was evaporated at a temperature of 30 C. to removewater and leave a residue of the corresponding urea which wasregenerated into the carbodiimide following the procedure of Amiard, G.and Heymes, R., Bull. Soc. Chim. France, 1360 (1956).

EXAMPLE 19 Phrhaloyl-L-Threonine To a solution of 4.54 g. (0.0306 mole)phthalic anhydride in 25 ml. of dry dioxane, there was added 3.04 g.(0.0255 mole) of L threonine. The heterogeneous mixture was submerged ina oil bath at 105 C. and stirred vigorously for 5 hours. Afterevaporation of the solvent under reduced pressure, thephthaloyl-L-threonine crystallized from acetone-water; yield, 6.0 g.(94%), M.P. 134140 C. A portion of the product was recrystallized twicefrom ethanol water, M.P. 143-144" C., [a] 36.7 (0.0480 g. in 1.5 ml.absolute eth anol).

Analysis-Gabi for C12H11NO5I C, H, N, 5.61. Found: C, 58.06; H, 4.75; N,5.54.

Phthaloyl-L-Threonyl-L-Phenylalanine M ethyl Ester To a solution of0.350 g. (0.0014 mole) of phtha loyl- L-threonine in 6 ml. of purifiedmethylene chloride, there was added freshly prepared L-phenylalaninemethyl ester (0. 500 g., 0.0028 rnole) (prepared by neutralizing anaqueous solution of L-phenylalanine methyl ester hydrochloride to pH 9.0with potassium carbonate and subse-' quent extraction into ether; thehydrochloride was prepared by Fischer esterification) and N,Ndicyclohexylcarbodiimide E. Schmidt, F. Hitzler and E. Lahde, Ber., 71,1933 (1938) (0.290 g. 0.0014 mole). A precipitate ofN,N-dicyclohexylurea formed immediately, but the reaction was allowed toproceed for 5 hours at room temperature. I

The urea was removed by filtration and the methylene chloride solutionwas extracted successively with N hydrochloric acid, N potassiumbicarbonate and water. After drying and subsequent removal of thesolvent, the residue was crystallized from acetone-ether, yielding atotal of 0.520 g. (91%) of product; M.P. l49-152 C. An analytical samplewas obtained after two recrystallizations from acetone-ether, M.P.153-154 C., +1.93 (0.0986 g. in 1.5 ml. of dimethylformamide).

Analysis,Calcd. for C H N O C, 64.38; H, 5.40; N, 6.83. Found: C, 64.63;H, 5.65; N, 7.11.

EXAMPLE 20 Phthalay[L-Threonyl-DLeucine Benzyl Ester To a solution of0.600 g. (0.00241 mole) of phthaloyl- L-threonine in ml. of distilledacetonitrile, there was added L-leucine benzyl ester prepared byneutralizing an aqueous solution of L-leucine benzyl ester hydrochlorideto pH 9.0 with potassium carbonate and subsequent extraction into ether.The hydrochloride was synthesized according to the procedure of H. K.Miller and H. Wa'elsch, J. Am. Chem. Soc, 74, 1092 (1952) (0.810 g.,0.0036 mole) and N,Ndicyclohexylcarbodiimide (0.495 g., 0.00241 mole).

The reaction was carried out as described forphthaloyl-L-threonyl-L-phenylalanine methyl ester. After theacetonitrile was removed by distillation under reduced pressure, theresidue was taken up in ethyl acetate and extracted with acid,bicarbonate and water. After drying and removal of the solvent theresidue was crystallized from acetonitrile-water yielding 0.980 g.(89%); M.P. 103106 C. Two recrystallizations from acetoneether-hexaneyielded an analytical sample; M.P. 109- 110 C., [111 -16.7 (0.0770 g. in1.5 ml. absolute ethanol).

AnalySis.'Calcd. for C25H29N206; C, H, N, 6.19. Found: C, 66.14; H,6.25; N, 6.32.

EXAMPLE 21 Phthaloyl-L-Threonyl-L-Phenylalanine To a solution of 0.840g. (0.00205 mole) of phthaloyl- L-threonyl-L-phenylalanine methyl esterin 30 ml. of acetone there was added 10 ml. of water and 5 ml. ofconcentrated hydrochloric acid. The solution was refluxed for two and ahalf hours and the acetone removed by distillation. Ethyl acetate wasadded and the product was subsequently extracted into bicarbonatesolution. After acidification and re-extraction into ethyl acetate,drying and removal of the solvent under reduced pressure, the productwas obtained as a crystalline mass. Recrystallization from ethanol-Wateryielded 0.510 g. (54%) of analytically pure material; M.P. 207-208 C.,'[a] +23.2 (0.025 g. in 1.5 ml. absolute ethanol).

Analysis.-Calcd. for C H N O C, 63.60; H, 5.09; N, 7.07. Found: C,63.30; H, 5.16; N, 7.13.

Phthaloyl-L-Threonyl-L-Phenylalanyl-L-Phenylalanine Methyl Ester Amixture of 0.200 g. (0.00051 mole) of phthaloyl-L-threonyl-L-phenylalanine, L-phenylalanine methyl ester (0.110 g.,0.00062 mole) and N,N-dicyclohexylcarbodiimide (0.105 g., 0.00051 mole)in methylene chloride was allowed to react as described forphthaloyl-L-threonyl-L- phenylalanine methyl ester.

The recrystallized product (ethanol-water) amounted to 0.245 g. (92%);M.P. 147.2-148" C.; 0 25.6 (0.0271 g. in 1.5 ml. of absolute ethanol).

Analysis.-Calcd. for C H N O C, 66.77; H, 5.60; N, 7.54. Found: C,66.71; H, 5.86; N, 7.48.

EXAMPLE 22 O-Acetyl-L-Serine A solution of 3.5 g. (0.0286 mole) ofL-serine in 150 ml. of glacial acetic acid was saturated with hydrogenchloride at 0 C. The mixture was stored for fifteen hours at roomtemperature, and the solvent removed under reduced pressure. Afterrepetition of this process, the O-acetyl-L-serine hydrochloride wascrystallized from ethanol-ether, yielding 5.20 g. (99%); d. 160 'C., 13-7.4 (0.0334 g. in 1.5 ml. absolute ethanol). To a solution of 3.0 g.(0.0163 mole) of this product in ethanol there was added 1.62 g. (0.0160mole) of triethylamine. The resulting O-acetyl-L-serine wasrecrystallized from water-ethanol,'2.42 g. (98%); d. 167-168 C., 0 +9.15(0.0268 g. in 1.5 ml. 0.1 N hydrochloric acid).

A naZysis.Calcd. for C H NO C, 40.82; H, 6.12; N, 9.51. Found: C, 40.92;H, 6.41; N, 9.39.

Phthaloyl-O-Acetyl-L-Serine To a solution of phthalic anhydride (2.0 g.,0.0135 mole) in 20 ml. of dry dioxane, there was added 1.65 g. (0.0112mole) of O-acetyl-L-serine. The heterogeneous mixture was submerged inan oil bath at C. and stirred vigorously for 12 hours. The dioxanesolution was lyophilized. The resulting oil was taken up inetherchloroform mixture (3:1) and filtered. The solution waschromatographed on a g. of silica gel column (Davison-Silica-gel, lotNo. 923-08-08-226, 100-200 mesh), previously washed with ether, thenwith ether-chloroform (3:1). The crystalline product 1.93 g. (63%)emerged with the solvent front; M.P. -147 C. A portion of 11 thismaterial was recrystallized from ethanol-water for analysis; M.P.151-153 C., [u] D 63.1 (0.0363 g. in 1.5 ml. absolute ethanol).

Analysis.Calcd. for 'C H NO C, 56.32; H, 4.00; N, 5.05. Found: C, 56.39;H, 3.85; N, 5.04.

PhthaloyI-O-Acetyl-L-Seryl-L-Phenylalahine Methyl Ester A methylenechloride solution of 0.400 g. (0.00145 mole) ofphthaloyl-O-acetyl-L-serine, L-phenylalanine methyl ester (0.310 g.,0.00165 mole) and N,N'-dicyclohexylcarbodiimide (0.300 g., 0.0014 mole)was allowed to react as described forphthaloyl-L-threonyl-L-phenylalanine methyl ester.

Recrystallization from ethanol-water afforded a total of 0.570 g. (89%);M.P. 131-132 C., [04 1) +14.7 (0.0395 g. in 1.5 ml. of absoluteethanol).

Analysis.Calcd. for C H N O C, 63.01; H, 5.06; N, 6.39. Found: C, 63.21;H, 5.34; N, 6.44.

EXAMPLE 23 Carbobenzoxy-L-Hydroxyprlyl-L-Phenylalanine Methyl Ester Asolution of carbobenzoxy-L-hydroxypnoline J. S. Fruton, J. Biol. Chem.,146, 463 (1942) (0.8 g., 0.0032 mole), Lphenylalanine methyl ester (0.54g., 0.0032 mole), and N,N-dicyclohexylcarbodiimide (0.625 g., 0.0033mole) in methylene chloride was treated as dcscribed forphthaloyl-L-threonyl-L-phenylalanine methyl ester. Crystallizationoccurred from an ethyl acetate solution; yield, 1.1 g. (86%), M.P.108112 C. A portion of the material was recrystallized fromacetone-etherhexane; M.P. 114-115 C., [a] D --29.2 (0.0416 g. in 1.5 ml.absolute ethanol).

=Analysis.Calcd. for C H N O C, 64.78; H, 6.10; N, 6.57. Found: C,64.76; H, 6.36; N, 6.60.

EXAMPLE 24 Phthaloylglycylglycine Ethyl Ester Run (a).To a suspension of0.21 g. (1.46 mmole) of glycine ethyl ester hydrochloride in 4 ml. ofdry dioxane was added 0.38 g. (1.56 mmole) of 1-cyclyhexyl-3-[2-morpholinyl-(4)-ethyl] carbodiimide followed by 0.36 g. (1.46 mole)of phthaloylglycine. The mixture was stirred overnight after which thedioxane was distilled under reduced pressure. The residue was dissolvedin ethyl acetate and this solution was washed first with 6 ml. of 1 Nhydrochloric acid followed by 6 ml. of 1 N potassium bicarbonate. Theethyl acetate layer was dried over magnesium sulfate then evaporated todryness. The residue, after crystallization from ethanol, weighed 0.33g. (80% M.P. 191193 C. A mixed melting point with authentic material wasnot depressed.

Run (b).-The procedure given above was followed using a mixture of 5 ml.of dioxane and 1 ml. of water as the solvent. The other quantities were:0.21 g. (1.46 mmole) of glycine ethyl ester hydrochloride, 0.43 g. (1.56 mmole) of N-cyclohexyl-N'-(4-diethylaminocyclohexyl) carbodiimide and0.3 g. (1.46 mmole) of phthaloylglycine. The product weighed 0.24 g.(83% M. P. 193-195 C.

Run (c).--To a solution of 0.18 g. (0.865 mmole) of phthaloylglycine'and 0.088 g. (0.865 mmole) of glycine ethyl ester in 6 ml. of water wasadded 0.4 g. (0.86 mmole) of the methyl p-toluenesulfonate salt ofN-cyclo hexyl-N'-(4-diethylaminocyclohexyl) carbodiimide. A solid beganto separate after 10 minutes of stirring and after 2 hours the solid wascollected, weight 0.18 g. (75%); M.P. 194195 C.

Run (d).The procedure given in Run (a) was followed using dioxane as thesolvent, 24.7 mg. (0.29 mmole) of glycine ethyl ester hydrochloride,59.0 mg. (0.29 mmole) of phthaloylglycine and 0.1 g. (0.29 mmole) ofl,3-di-(4-diethylaminocyclohexyl) carbodiimide. The yield was 50 mg.(86%); M.P. 192194 C.

12 EXAMPLE 2s Phthaloyl-L-Phenylalanylglycine Ethyl Ester Run (a).-Theexperimental procedure described above for the preparation ofphthaloylglycylglycine [Example 24, Run (a)] was followed using 0.196 g.(1.4 mmole) of glycine ethyl ester hydrochloride, 0.4 g. (1.7 mmole) ofN cyclohexyl N'-[2-morpholinyl-( 4) -ethy1] carbodiimide and 0.415 g.(1.4 mmole) of phth-aloyl-L-phenylalanine. Recrystallization of theproduct from ethanol yielded 0.43 g. (81%); M.P. 160161 C., [ad n 146ethanol. The product corresponded in rotation and melting point with anauthentic sample [J C. Sheehan, D. W. Chapman and Roy W. Roth, 1. Am.Chem. Soc., 74, 3822 (1952)].

Run (b).To a solution of 0.196 g. (1.4 mmole) of glycine ethyl esterhydrochloric and 0.41 g. (1.5 mmole) ofN-cyclohexyl-N-(4)-diethylaminocyclohexyl carbodiimide in 6 ml. ofdioxane was added, with stirring, 0.45 g. (1.4 mmole) ofphthaloyl-L-phenylalanine. The resulting reaction mixture was worked upas described above and afforded 0.49 g. M.P. -161 C.; [M 15 146[ethanol].

Run (c).--The reactant amounts given in Run (b) were used along with amixture of 2 ml. of water and 6 ml. of dioxane as the solvent. Theproduct weighed 3.39 g. (71%); M.P. 160161 C., [a] D 146 [ethanol].

Run (d) .This experiment was carried out as described in Run (a) usingdioxane, 0.196 g. (1.4 mmole) of glycine ethyl ester hydrochloride, 0.38g. (1.7 mmole) of N- cyclohexyl N (fi-diethylaminoethyl) carbodiimideand 0.415 g. (1.4 mmole) of phthaloyl-L-phenylalanine. Recrystallizationfrom ethanol yielded 0.11 g. (20%) of product; M.P. 160-161 C.; [a] D--146 [ethanol].

EXAMPLE 26 Phthaloyl-L-PhenylalanyI-Leucine Ethyl Ester Run (a) Theexperimental procedure described for phthaloyl-L-glycylglycine ethylester [Run (a)] was followed using 0.27 g. (1.36 mole) of L-leucineethyl ester hydrochloride, 5 m1. of dioxane, 0.39 g. (1.4 mmole) ofN-cyelohexyl N (4) diethylaminocyclohexyl carbodiimide and 0.4 g. 1.36mmole) of phthaloyl-L-phenylal-anine; yield, 0.5 g. (87%); M.P. 108-109C.; [(11 1) 1 15 [ethanol]. The mixed melting point with an authenticsample did not show depression.

Run (b).-This process was carried out as above with the variation that 2cc. of water was added to the initial reaction mixture; yield, 50%

EXAMPLE 27 Carbobenzoxyglycyl-L-Phenylalanylglycine Ethyl Ester Theexperimental procedure given in Run (a) above was followed using 0.12 g.(0.85 mmole) of glycine ethyl ester hydrochloride, 3 ml. of dioxane,0.24 g. (0.85 mmole) of 1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide and 0.3 g. (0.85 mmole) ofcarbobenzoxyglycyLL-phenylalanine; yield, 0.3 g. (81%); M.P. 117- 118C.; [a] D --12.4 [ethanol] (reported: M.P. 116- 118 C., [@1 0 12).

EXAMPLE 28 Phthaloyl-L-Phenylalanyl-L-Phenylalanine Methyl Ester 13EXAMPLE 29 Phenylacetanilide Via the Quaternary Carbodiimide, 1-

Ethyl 3 [2 Morpholinyl (4) Ethyl] Carbodiimide M etho-p-ToluenesulfhateA solution of 0.184 g. (1.35 mmoles) 0f phenylacetic acid, 0.126 g.(1.35 mmoles) of aniline, and 0.50 g. (1.35 mmoles) of quarternarycarbodiimide, 1-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemetho-p-toluenesulfonate, in 25 ml. of purified methylene chloride washeated under reflux Overnight. A small quantity of the urea which hadseparated was removed by filtration (M.P. 182183) and the filtratewashed successively with ml. portions of water, N hydrochloric acid, andN sodium bicarbonate. The dried (magnesium sulfate) solution wasconcentrated (aspirator) to yield 0.238 g. (83%) of phenylacetanilide,M.P. 116-118 C. The product was recrystallized from benzene-petroleumether (B.P. 30- 60 C.) to give colorless needles melting at 117.8- 118.0C.

EXAMPLE 30 P henylacetanilide Via Dicyclohexylcarbodiimide To a stirredsolution of 0.33 g. (2.42 mmoles) of phenylacetic acid and 0.225 g.(2.42 mmoles) of aniline in 25 ml. of benzene was added 0.50 g. (2.42moles) of N,N-- dicyclohexylcarbodiimide. The dicyclohexylurea formed inthe reaction started to separate within a few seconds. After stirringovernight the urea was removed by filtration (M.P. 234.5235.5 C.) andthe filtrate washed successively with 10 ml. portions of N sodiumbicarbonate and N hydrochloric acid. After drying over magnesium sulfatethe solution was evaporated under reduced pressure. Crystallization ofthe residue from acetone cyclohexane gave 0.357 g. (70%) ofphenylacetanilide, M.P. ll8.0118.5 C.

EXAMPLE 31 N -Formy l-e-N aphthy [amine A solution of 0.194 g. (1.35moles) of ,B-naphthylamine, 0.062 g. (1.35 mmoles) of anhydrous formicacid, and 0.50 g. (1.35 mmoles) of the quaternary carbodiimide,1-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemetho-p-toluene-sulfonate, in 20 ml. of purified methylene chloride washeated under reflux for 4 hours. The resulting pin-lg solution waswashed successively with 10 ml. of water, N hydrochloric acid and Nsodium bicarbonate. Concentration afforded a crystalline residue, 0.190g. (82%), M.P. 127.5-l28.5 C. Recrystallization from methylenechloride-petroleum ether (B.P. 30-50 C.) gave colorless plateletsmelting at 129.5130.5 C. (lit. M.P. 129 C.). 7

EXAMPLE 32 Trifluoroacetanilide A solution of 0.155 g. (1.35 moles) oftrifluoroacetic acid, 0.126 \g. (1.35 mmoles) of aniline, and 0.50 g. ofthe quaternary carbodiimid'e, 1-ethyl-3-[2-morpholinyl- (4)-ethyl]carbodiirnide metho-p-toluenesulfonate, in 25 ml. of purified methylenechloride was heated to reflux. After approximately 30 minutes the ureaformed in the reaction started to separate. After 6 hours of reflux themixture was cooled, the urea removed by filtration (M.P. 175-1785 C.),and the filtrate washed with 10 ml. of water and 10 ml. of Nhydrochloric acid. From the organic layer there was obtained acrystalline residue which was crystallized again from iso-octane. Theyield was 0.215 g. (85%), M.P. 90.5-91.0 C.

V Analysis.-Calcd. for C H F NO: C, 51.07; H, 3.22. Found: C, 51.72; H,3.45.

EXAMPLE 33 Benzanilide A solution of 0.165 g. (1.35 moles) of aenzorcacid,

0.126 g'. (1.35 mrnoles) of aniline, and 0.50 g. (1.35 moles) ofquaternary carbodiimide, l-ethyl-3-[2rnorpholinyl-(4) ethyl]carbodiimide metho-p-toluenesulfohate, in 20 m1. of purified methylenechloride was heated under reflux for 15 hours. The resulting colorlesssolution was washed with successive portions of water, N hydrochloricacid and N sodium bicarbonate, and then concentrated (aspirator) to acrystalline residue; 0.135 g. (51%), M.P. 164-165 C.

EXAMPLE 34 N [2-M orpholiny l- (4 -Ethyl Phenylacet'am ideAminoethylmorpholine (0.63 g.; 4.84 rnmoles) was added to a solution ofphenylacetic acid (0.66 g.; 4.84 mmoles) in 25 ml. of purifiedacetonitri-le. In approximately 3 minutes the salt,morpholinoethylammonium phenylacetate, separated from solution. Themixture was heated (oil bath) with stirring to 50 C., at whichtemperature the salt redissolved. Dicyclohexylcarbodiimide (1.0 g; 4.84mmoles) was then added. Within 1 minute the dicyclohexylurea formed inthe reaction started to separate from solution. Stirring was continuedat this temperature for 13 hours. The urea was removed by filtration(M.P. 234-236 C.) and the filtrate concentrated to a yellow oil. Thiswas dissolved in 15 ml. of benzene and the solution extracted with three10 ml. portions of N hydrochloric acid. Solid sodium bicarbonate wasadded to the aqueous extracts till basic (ca. pH 8) and the resultantsolution extracted with three 25 m1. portions of methylene chloride. Thedried organic layer was concentrated under reduced pressure to apartially crystalline residue. Crystallization from acetone-water gave0.207 g. (58%) of the amide, M.P. 82.5-84 C. A sample recrystallized foranalysis from acetone-water melted at 82.583.5 C.

Analysis.Calcd. for C H N O C, 67.71; H, 8.12. Found: C, 67.86; H, 8.29.

EXAMPLE 35 N-Cyclohexylphenylacetamide Via the Quaternary Carbodiimide,l-Ethyl-S-[2-M0rph0linyl-(4)-Ethyl] CarbodiimideMetho-p-Toluenesalfonate A solution of 0.203 g. (1.49 mmoles) ofphenylacetic acid, 0.148 g. (1.49 mmol'es) of cyclohexylamine, and 0.55g. 1.49 moles) of quaternary carbodi-imide, 1-ethyl-3-[2-morpholinyl-(4) ethyl] carbodiimide metho-ptoluenesulfonate,in 50 ml. of purified methylene chloride was heated under reflux for 12hours. The resulting colorless solution was concentrated (asp-irator) toa volume of 20 ml. and washed successively with 10 ml. portions ofwater, N hydrochloric acid, and N sodium bicarbonate. The dried(magnesium sulfate) solution was concentrated under reduced pressure andthe crystalline residue recrystallized from b enzene-iso-octane. Theyield was 0.10 g. (31%), M.P. 138.0-139.5 C.

EXAMPLE 36 N-Cyclohe xylphenylacetamide Via the Basic Carbodiimide,1-Ethyl-3-[2-M0rph0linyl (4) -Ethyl] Carbodiimide To a solution of 0.38g. (2.79 moles) of phenylacetic acid in 50 ml. of purified methylenechloride was added 0.277 g. (2.79 moles) of cyclohexylamine. The salt,cyclohexylammonium phenylacetate, separated immediately. The basiccarbodiimide, l-ethyl-3-[2-morpholinyl- (4)-ethyl] carbodiimide, (0.512g.; 2.79 mmoles) was added and the mixture heated under reflux for 9hours. Afiter an additional 48 hours at room temperature (stirring), themixture was cooled and the unreacted salt removed by filtration mg;12%). The filtrate was concentrated (.aspirator) to dryness and theresidue extracted with benzene. An additional 55 mg. (8.5%) of unreactedsalt remained. The benzene solution was washed successively with two 10ml. portions of N hydrochloric acid and 10 trnl. of sodium bicarbonate.The dried (magnesium sulfate) solution was concentrated under reducedpressure to a crystalline residue which was recrystallized :frombenzene-petroleum ether (B.P. 30-60" C.) to give 0.150 g. (25%) ofN-cyclohexylphenylacetamide, M.P. 137138 C.

EXAMPLE 37 N-Cyclohexylbenzamide Via Dicyclohexylcarbodiimide in AcetoneCyclohexylamine (0.24 g.; 2.42 mmoles) was added to a solution ofbenzoic acid (0.295 g.; 2.42 mmoles) in 25 ml. of acetone. The saltcyclohexylammonium benzoate separated immediately.Dicyclohexylcarbodiimide (0.50 g.; 2.42 mmoles) was added and themixture stirred at room temperature for 13 hours. Nearly all of thematerial dissolved. The solvent was removed under reduced pressure. Abenzene solution of the residue was concentrated gradually to give twocrops, a total of 0.30 g. (38%), of N-benz-oyl-N,N'-dicyclol1exylurea,M.P. 166- 167 C. A sample for analysis recrystallized frombenzene-petroleum ether (B.P. 30-60 C.), melted at 166.0- 166.5 C.

Analysis.-Calcd. for C H N O C, 73.13; H, 8.59. Found: C, 73.02; H,8.68.

The combined filtrates (above) were washed successively with 5 ml. of Nbicarbonate and tWo 5 ml. portions of N hydrochloric acid. Petroleumether was added gradually to the dried (potassium carbonate) benzenesolution. An additional 70 mg. (9%) of acylurea (colorless needles) and69 mg. (14%) of N-cyclohexylbenzamide (platelets), M.P. 149-150 C., wereisolated.

EXAMPLE 3 8 N-C'yclohexylbenzamide Via the Quaternary Carbodiimide,1-Ethyl-3-[2-M0rph0linyl-(4)-Ethyl] CarbodiimideMetho-p-Tolaenesulfonate A solution of 0.334 g. (2.74 mmoles) of benzoicacid, 0.277 g. (2.79 mmoles) of cycloheXyla-mine, and 0.512 mmoles ofthe quaternary carbodiimide, 1-ethyl-3-[2- morpholinyl (4)-ethyl]carbodiimide metho-p-toluenesulfonate, in 20 m1. of purified methylenechloride was stirred at room temperature for 10 hours. One drop ofglacial acetic acid was added to decompose any unreacted car bodiimideand after standing an additional 15 minutes the solution wasconcentrated (aspirator) to dryness. After extraction of the residuewith benzene there remained 0.510 g. (83.5% recovery) of unreactedcyclohexylammonium benzoate, M.P. 183-185 C. The benzene solution waswashed with three 10 ml. portions of N hydrochloric acid and 10 ml. of Nsodium bicarbonate. The dried (magnesium sulfate) solution wasevaporated under reduced pressure and the crystalline residuerecrystallized from benzene-iso-octane. The yield was 50 mg. (9%) ofN-cyclohexylbenzamide melting at 147-149 C.

EXAMPLE 39 Carbodiimide percent of gel weight: Melting point, "C 33.0 2.33.0 34.9 10 52.5 25 Over 90.0

1 6 EXAMPLE 40 Phenylacetanilide Via 1-Ethyl-3-Phenylcarbodiimide 1 To asolution of 0.33 g. of phenylacetic acid and 0.225 g. of aniline in 25ml. purified methylene chloride was added 0.5 g. of1-ethyl-3-phenylcarbodiimide. After storage for 48 hours at 25 C. therewas isolated phenylacteanilide, M.P. 116-118 C.

EXAMPLE 41 Treatment of Collagen With a Water-Soluble Carbodiimide Apresoaked (in distilled water) fiber of kangaroo tail tendon wasimmerse-d for 24 hours at 25 C. in 10% aqueous solution of1-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemetho-p-toluenesulfonate.

The treated fiber shrank to a much less extent than did an untreatedfiber when immersed in water at 90 C.

EXAMPLE 42 To a solution of 2.0 g. of gelatin in 25.0 ml. of water wasadded 1.5 g, of 1-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemetho-p-toluenesulfonate. The solution gelled almost immediately (30seconds), but liquefied on storage at room temperature for 16 hours. Theresulting solution was dialyzed (using cellulose casing) for 4 days. Thesolution was freeze-dried to a white powder; weight 2.0 g. A Sorensentitration (G. Wilson, Laboratory Manual of Physiological Chemistry, TheWilliams and Wilkins Co., Baltimore, Md., 1952, p. 72) on 0.4 g.required 0.25 ml. of 0.103 N sodium hydroxide. An untreated gelatinsample (0.4 g.) required 2.6 ml. A Van Slyke analysis yielded 0.19%nitrogen, while untreated gelatin yielded 0.64% nitrogen.

EXAMPLE 43 A solution of 4.0 g. of gelatin in 50 ml. of water was gelledby mixing with 2.25 g. of 1-ethyl-3-[2-morpholinyl- (4)-ethyl]carbodiimi-de metho-p-toluenesulfonate. The gel was immediatelyfreeze-dried, followed by trituration with 500 ml. of water. Theresulting material after drying weighed 3.0 g. The wash Water onevaporation yielded 2.3 g. of 1-ethyl-3[2-morpholinyl-(4)-ethyl] ureametho-p-toluenesulfonate.

A 0.5 g. sample of the above-treated gelatin was dissolved in 20 ml. ofa 50% calcium chloride solution by heating on a steam bath for 1 hour..A Sorensen titration required 0.65 ml. of 0.103 N sodium hydroxide.

When 0.1 g. of the gelatin prepared above was suspended in 2.0 ml. ofwater containing 75 mg. of 1-ethyl-3- [2-morpholinyl-(4) -ethyl] ureametho-patoluenesulfonate, no liquefaction occurred even after 9 days.If, however, the carbodiirnide was substituted for the urea,liquefaction occurred.

When conductometric amino titrations S. Ellis and I. P arkhurst,Biochem. J 52, 350 (1952), were carried out on the gelatin products of adecrease in amino content as compared to untreated gelatin was observed.

EXAMPLE 44 A solution of 0.2 g. of gelatin, 0.45 g. of urea and 0.15 g.of 1-ethyl-3-[2-morpholinyl-(4) ethyl] carbodiimidemetho-p-toluenesulfonate in 4.0 ml. of water gelled after 20 seconds. Onstorage overnight the reaction mixture liquefied.

EXAMPLE 45 To a solution of 0.1 g. of benzoyl gelatin in 2 ml. of waterwas added 0.1 g. of 1-ethyl-[2-morpholinyl-(4)- ethyl] carbodiimidemetho-p-toluenesulfonate. On stirring no sudden gelation occurred aswith the control gelatin.

1 Weitll, Bern, 8, 1530 (I875).

17 EXAMPLE 46 The liquefied, dialyzed, and then freeze-dried gelatin ofExample 42 was redissolved in water to produce a solution. This solutionwas useful as a plasma substitute or blood plasma extender. It is devoidof amino acids other than those characteristic of gelatin. This newproduct permits the ultiliz-ation of higher amino acid or proteincontent of gelatin in injectable form without the danger of gelationduring or after injection. Thus, higher nutrient :and plasma simulatingvalues are possessed by the instant carbodiimide treated gelatin. Notonly is this product a satisfactory substitute for blood plasma but byreason of the fact that it may be safely introduced into the body inlarge quantities it, as well as the other gelatin products of thisinvention, may be employed as a vehicle for the introduction byinjection or orally of many medicines and foods, for example,antibiotics (e.g., penicillins, tetracyclines), insulin, glucose, orprotein hydrolysates containing essential amino acids. The gelatinproducts of this invention, and of this example are useful in place ofthe oxypolygelatins as illustrated in Patents 2,591,133 and 2,811,482.

EXAMPLE 47 The condensing action of carbodiimides upon gelatin toproduce an insoluble gelatin is useful in the production of gelatincoated products having tough exteriors, suitable as enteric coatings,and preventing adhesion or agglomeration of the individual tablets,capsules, or if desired, microspheres.

Aspirin tablets were quickly dipped in a 5% aqueous gelatin solution andthen allowed to dry. The aspirin tablets thus coated with gelatin werethen passed through an aqueous solution of 1-ethyl-3-[2-morpholinyl-(4)-e-thy1] carbodiimide metho-p-toluenesulfonate, removed, quicklywashed with water, and allowed to dry. The tablets were firm, non-tacky,with improved structural strengm, not readily disintegratable in water,but readily soluble in the intestine.

EXAMPLE 48 Soft gelatin capsules containing vitamins were passed througha 10% aqueous solution of 1-ethyl-3-[2-morpholinyl-(4)-ethyl]carbodiimide metho-p-toluenesulfonate, washed with water, and thendried. The capsules were non-adherent in the presence of atmosphericmoisture and readily disintegrated upon oral administration. Thesecapsules did not require treatment with lacquer to preventagglomeration. In contrast to formaldehydetreated gelatin which producederratic enteric coatings that sometimes disintegrated in the stomach andat other times did not disintegrate at all and always became moreinsoluble upon standing, gelatin treated with carbodiimide in accordancewith this procedure produced highly uniform capsules and gelatincoatings possessed of unvaryin g properties not changing with age.

EXAMPLE 49 In the manufacture of soft gelatin capsules, the gelatinsheets were formulated with carbodiimide treated gelatin having amelting point of about 50 0, made as described in Example .39,plasticized with glycerine. The resulting soft capsules were moistureresistant.

EXAMPLE 5 O A 10% aqueous solution of carbodiimide treated gelatin,prepared 13.8 in Example 39, first paragraph, was whipped and aerated toa uniform foam and baked at 250 F. to dehydrate completely and sterilizethe prodnot. The carbodiimide gelatin foam was useful as a surgicalsponge. It had low-water disintegrability proper-ties and remaineduniform upon long storage. It was eventually absorbed by body fluidswhen enclosed in an animal body cavity.

18 EXAMPLE 51 A surgical sheep-gut suture was immersed in a. 10% aqueouscarbodiimide, 1-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemethyl-p-toluene sulfonate solution for 6 hours, and washed with water.It was found to be pliable but with a higher tensile strength thanoriginally possessed by the untreated suture material.

EXAMPLE 52 A wet, developed photographic silver emulsion negative filmbearing a gelatin base emulsion on a cellulose acetate backing wasdipped into a 5% aqueous carbodiimide, -1- cyclohexyl-3 (4diethylaminocyclohexyl) carbodiimide metho-p-toluenesulfonate, for 5minutes, removed, and washed with water. Even before drying, thehardening action upon the gelatin was noticeable. After drying, thehardness of the gelatin was prevailed in comparison with untreatedgelatin emulsion films.

EXAMPLE 53 A sample of woolen worsted cloth was immersed for 24 hours ina 5% aqueous solution of 1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide metho-p-toluenesulfonate, removed, rinsed with water anddried. Upon testing this treated fabric for shrinkage by washing it inwater, it was found to exhibit desirable reduced shrinkage as comparedwith an untreated control. It was also found to have improved feel, adesirable attribute of wool cloth.

EXAMPLE 54 Otherwise like Example 53, using a silk cloth sample in placeof the woolen worsted produced an improved silk having good lustre andcrease resistance.

EXAMPLE 55 A fur felt was immersed for 24 hours in a 5% aqueous solutionof l-cyclohexyl 3 (4-diethylaminocyclohexyl) carbodiimidemetho-p-toluenesulfonate, removed, washed with water and dried. Thisfelt showed improved dimensional stability upon repeated wetting anddrying as compared with untreated felt.

EXAMPLE 56 The methods of Examples 53 and 55 were modified to the extentof employing a pretreatment of the polypeptide material with 1% aqueouspapain for 6 hours at a pH of 7.5, or, alternatively, a pretreatmentwith trisodium phosphate at pH 12 for 6 hours, before the carbodiimidetreatment described in Examples 53 and 55 was carried out. Thesepretreatments were found especially effective.

EXAMPLE 57 Chicken body feathers were washed in water and curled bydrying and drawing over sharply curved surfaces. The curled featherswere treated with an organic solution of N,N-dicyclohexylcarbodiimide,10% in benzene, in excess. The resulting feathers showed increasedwater-repellency and improved flulfiness retention.

EXAMPLE 58 Fine womans hair, which had never been previously waved, waswashed, dried and then wetted with a 5% aqueous solution ofl-ethyl-3-[2-morpholinyl-(4)-ethyl] carbodiimidemetho-p-toluenesulfonate, portions of the hair were wound on plasticmandrels, using absorbent end papers, under tension and the hair waspermitted to remain for 3 hours on the mandrels. Upon removing the endpapers and mandrels, it was found that a firm curl with unimpaired hairgloss and texture resulted.

While the present invention has been described with reference tospecific details and examples it should not be construed as limitedthereby but instead reference should be had to the appended claims for adefinition of the scope of the invention.

I claim:

1. The method of improving the shrink and crease resistance of a fabriccomposed of fibers consisting essentially of a proteinaceous substanceselected from the group consisting of wool and silk which comprisesimmersing said fabric in an aqueous solution of a water-soluble organicmonocarbodiimide having the formula:

wherein one of the monovalent radicals R and R is an aliphatic radicaland the remaining of said radicals is a radical selected from the groupconsisting of aliphatic and aromatic radicals, removing said fabric fromsaid solution, washing said fabric with water and drying said fabric.

2. The method of claim 1 wherein said water-soluble, organicmonocarbodiimide is 1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide metho-p-toluene sulfonate.

3. The method of increasing the tensile strength of sheep-gut sutureswhich comprises immersing said sutures in an aqueous solution of awater-soluble, organic monocarbodiimide having the formula:

wherein one of the monovalent radicals R and R is an aliphatic radicaland the remaining of said radicals is a radical selected from the groupconsisting of aliphatic and aromatic radicals, removing said suturesfrom said solution, washing said sutures with water, and drying saidsutures.

4. The method of claim 3 wherein said water-soluble organicmonocarbodiimide is 1-ethyl-3-(2-morpholinyl- (4)-ethyl) carbodiimidemetho-p-toluene sulfonate.

5. The method of increasing the resistance of soft gelatin capsules todisintegration upon being :orally administered which comprises immersingsaid capsules in an aqueous solution of a water-soluble organicmonocarbodiimide having the formula:

wherein one of the monovalent radicals R and R is an aliphatic radicaland the remaining of said radicals is a radical selected from the groupconsisting of aliphatic and aromatic radicals, removing said capsulesfrom said solution, washing said capsules with water and drying saidcapsules.

6. The method of claim 5 wherein said water-soluble organicmonocarbodiimide is l-ethyl-3-(Z-morpholinyl- (4)-ethyl) carbodiimi-demetho-p-toluene sulfonate.

7. The method of increasing the resistance of soft gelatin coatingssurrounding a medicament to disintegration upon being orallyadministered which comprises immersing a medicament having an exposedcoating of soft gelatin in an aqueous solution of a water-solubleorganic monocarbodiimide having the formula:

wherein one of the monovalent radicals R and R is an aliphatic radicaland the remaining of said radicals is a radical selected from the groupconsisting of aliphatic and aromatic radicals, removing said gelatincoated medicament from said solution, washing said coated medicamentwith water and drying said coated medicament.

8. The method of claim 7 wherein said water-soluble organicmonocarbodiimide is 1-ethyl-3-(2-morpholinyl- (4) -ethyl) carbodiimidemetho-p-toluene sulfonate.

9. The method hardening the gelatin coating of a photographic negativefilm composed of a cellulose derivative base having a coating thereoncomposed of a gelatin emulsion containing light sensitive particleswhich comprises immersing the coated film in an aqueous solution 20' ofa water-soluble organic monocarbodiirnide having the formula:

R -N=C=NR wherein one of the rn-onovalent radical-s R and R is analiphatic radical and the remaining of said radicals is a radicalselected from the group consisting of aliphatic and aromatic radicals,removing said film from said solution, washing said film with water anddrying said film.

10. The method of claim 9 wherein said water-soluble organicmonocarbodiimide is 1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide metho p toluene sulfonate.

11. The process for imparting a permanent configuration to human hairwhich comprises imparting a desired configuration to human hair,immersing the configured hair in an aqueous solution of a water-solubleorganic monocarbodiimide having the formula:

wherein one of the monovalent radicals R and R is an aliphatic radicaland the remaining of said radicals is a radical selected from the groupconsisting of aliphatic and aromatic radicals, withdrawing theconfigured hair from said solution, washing the configured hair withwater and drying the configured hair.

12. The process of claim 11 wherein the water 'soluble organicmonocarbodiimide is 1-ethyl-3-(2-morpholinyl- (4)-ethyl) carbodiimidemetho-p-toluene sulfonate.

13. A therapeutic composition comprising a medicament surrounded by anenteric coating of gelatin obtained by immersing a medicament having anexposed coating of soft gelatin in an aqueous solution of awater-soluble organic monocarbodiimide having the formula wherein one ofthe monovalent radicals R and R is an aliphatic radical and theremaining of said radicals is a radical selected from the groupconsisting of aliphatic and aromatic radicals, removing said gelatincoated medicament from said solution, washing said coated medicamentwith water and drying said coated medicament, said coating of gelatinbeing resistant to disintegration upon oral administration.

14. The method of producing a surgical sponge which comprises mixing anaqueous solution of gelatin with a water-soluble organicmonocarbodiimide having the formula R --N=C=N R wherein one of themonovalent radicals R and R is an aliphatic radical and the remaining ofsaid radicals is a radical selected from the group consisting ofaliphatic and aromatic radicals, to obtain an aqueous solution ofcarbodiimide treated gelatin, aerating said solution of form a foamthereof and subsequently dehydrating said foam by heating at elevatedtemperatures.

15. A surgical sponge obtained by the method of claim 14.

References Cited in the file of this patent

1. THE METHOD OF IMPROVING THE SHRINK AND CREASE RESISTANCE OF A FABRICCOMPOSED OF FIBERS CONSISTING ESSENTIALLY OF A PROTEINACEOUS SUBSTANCESELECTED FROM THE GROUP CONSISTING OF WOOL AND SILK WHICH COMPRISESIMMERSING SAID FABRIC IN AN AQUEOUS SOLUTION OF A WATER-SOLUBLE ORGANICMONOCARBODIIMIDE HAVING THE FORMULA: